The present invention relates to a new method and system for non-contact collection of cells or other target regions following laser microdissection from tissue sections, tissue-derived cell preparations, other biologically-derived materials, or non-biological materials that have been mounted onto a specially-prepared slide.
In the field of life science, selecting and successfully collecting homogeneous cells of interest without contamination from heterogeneous tissue samples is a precondition for the accurate and specific isolation and characterization of biologically relevant molecules that pertain to the normal, diseased, or malignant state of the cell population.
The earliest technique for microdissection separation was using a glass needle or other tool to separate target regions mechanically from tissue sections, an operation achieved by hand or assisted by a manipulator. This method possessed many disadvantages, including a high degree of difficulty, high time consumption, low efficiency and precision, high demand for skills of the operator, and low bioactivity of separated biological samples caused by mechanical injury.
In 1996, the laser was introduced to precisely dissect microscopic target regions of interest. Since that time, laser microdissection systems have been used for dissection and separation in the biological and medical field. Laser microdissection systems are characterized by excellent effectiveness of dissection, high efficiency of collection, and timely completion; there are minor adverse effects on the subsequent analysis of the genes or proteins. So far, the techniques of separation and collection based on laser microdissection involve laser capture microdissection technique (LCM), laser pressure catapulting technique (LPC), sticky membrane transfer technique, and a collecting technique using the force of gravity. These distinct methods for collection are the core techniques of laser microdissection system.
The laser capture microdissection technique utilizes a low-power infrared laser to melt a special thermoplastic film over target regions of tissue sections (for example, Bear et al., Laser Capture Microdissection Method and Apparatus, PCT No. PCT/US98/02388). The melted film expands after absorbing the laser energy, and adheres to target regions underneath. When the film is moved away, target regions attached to the film are separated successfully from tissue sections. The main disadvantages of this technique include mechanical damage on biological samples during separation and low precision because the film may also stick cells around target regions.
The laser catapulting technique uses a laser beam to dissect target regions, followed by the laser being defocused to strike the glass which creates a photo-acoustic pressure wave that causes the dissected target regions to catapult, overcoming gravity, into a sample collector (for example, U.S. Pat. No. 5,998,129 to Schutze et al., Method and Device for the Contactless Laser Assisted Microinjection, Sorting and Production of Biological Objects Generated in a Planar Manner). Disadvantages for this technique include the additional time required to defocus the laser, then re-focus on the sample, and the possibility that the UV laser beam may strike the middle of the cells, where the DNA and RNA may be altered by the laser.
The sticky membrane transfer technique utilizes a special sticky membrane to stick dissected target regions and collect the sample after the dissection (for example, M. Böhm et al., Membrane-Based Laser Microdissection in Molecular Oncology, ONKOLOGIE, 2000; 22:296-301). The membrane used in this method may lead to higher cost.
Collecting by the force of gravity must be based on the upright microscope (Koelble et al., The Leica Microdissection System: Design and Applications, J. MOL MED, 78 (7): B24-25, 2000). The dissected target regions fall down by the force of gravity directly into a collecting container located beneath the objective lens and microscope slide (which has to be turned upside down to place it on the microscope stage before beginning the procedure).
There is a need for a method and apparatus to collect cells following laser microdissection that avoids the problems of the prior art. The present invention meets this need.